Dilution calculation - (Jul/31/2013 )

Dear Colleagues 

I have actually some technical questions :

 

1. I want any one of you to send me a simple protocol for Poly A adenylation of miRNA followed by RT reaction for cDNA synthesis. I will give u one , if any comment, let me know 

 

- polyadenylation in a reaction volum 20 µl  

Poly A polymerase E   1 µl (5 unite) 

Poly A polymerase E buffer (10 X) 2 µl 

RNA templet  (1 µl of 400 ng/µl)

ATPs ( 10 mM) 2 µl 

water  14 µl

 for 37 C for 10 min, 

 

 then I will measure again the RNA concentration to adjust it again to 400 ng / µl then I will prepare a RT reaction volum 20 µl 

 

DNTPsa  1 µl 

RT E   1 µl 

RT E  buffer 4 µl 

RNA templet 1µl 

oligo Dt primer 2 µl 

Water  11 µl 

mix the mixure, centrifuge it then 50 C for 30 min and 85 c for 5 min , 

 

Is this right or wrong ? 

 

 

 

 

2. I received a ATP as a stock of 10 mM and as clear from above I want to use 1 mM in the reaction volum 20µl. How can I calculate the volum that I need to be added from this information 

 

3. The Oligo dt primer that I received is 100 µM and I need about 100 picoM in the reaction volum 20 µl. How can I calculate the volum that I need to be added from this information ?  I think the same answer like above. 

 

4. Why there is a difference between primers used for q-PCR and normal PCR for the same gene ? I know that u have to dilute the primers 1 : 10 or 1 : 20 if used for q-PCR. 

 

Thanks alot 

 

 

 

Basic equations will work for you - C1V1=C2V2 where C1 and V1= concentration and volume before and, C2 and V2 =concentration and volume after.  Rearrange equation to find the variable you want to measure.

 

uM (micromol per litre) = picomol per microlitre...

 

I don't know of any physical difference between the primers, the dilution is surely a matter for detection/efficiency.

please, I want to ask. What is the meaning of 10 mM of ATP when written on the coming product (stock ) is this mean 10 mM per liter then if I want to calculate how volum I talk to have 1 mM  this should be like this : 

 

10 mM --- > 1000000 µl  means  10000 µM ------ 1000000 µl   means 1 µM -------- 100 µl means If I take 1 µl from the stock it will have 0.1 µM  but I need a working solution of 1 mM so I need to take 100 ml from the stock to get a 1 mM ? 

 

another option is to apply ur formula VXn = VXN 

so I will form a solution of 500 µl that have a conc of 1 mM 

so I will take 50 µl from stock and add to it 450 µl water to form a solution of 1 mM conc then I will take 1 µl from it ? is this is right 

10 mM per liter has no meaning. 10 mM means 10 millimolar, or 10 millimoles per liter. You are confusing molar with moles.  One of these is a concentration (molar) and has units of moles per liter. The other, moles, is an amount, and has units of moles. To make a 1 mM solution, you have to dilute a 10 mM solution by a factor of 10.